The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on determining the contribution to the development, regeneration, and malignant transformation of oral tissues. Cell surface serine proteases as regulators of epithelial development, homeostasis regeneration, and malignancy Background: Cell behavior in higher eukaryotes is regulated by a large number of proteases and protease inhibitors that operate in the pericellular environment to provide focal proteolysis that is essential for cytokine/growth factor maturation, matrix remodeling, signaling receptor activation and shedding, ion channel activity, and more. Research accomplished: Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy Congenital tufting enteropathy (CTE) is a genetic disorder with a recessive Mendelian inheritance. It presents at birth as a severe intestinal insufficiency associated with watery diarrhea, dehydration, and failure to thrive in the absence of parenteral feeding. Histologically, CTE is characterized by epithelial dysplasia, villous atrophy and a compromised intestinal epithelial barrier. In 70% of patients, the underlying mutation is in the EPCAM gene, encoding the epithelial cell adhesion molecule (EpCAM). Interestingly, mutations in the SPINT2 gene, encoding the transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor (HAI)-2, have recently been described as a second cause of CTE, with EPCAM and SPINT2 mutations being mutually exclusive. In the absence of HAI-2, the membrane-anchored serine protease, matriptase, has been shown to cleave EpCAM in cultured intestinal epithelial cells, triggering the premature degradation of the tight junction protein claudin-7. Null mutations in mouse Spint2 causes early embryonic lethality. We found, however, that this embryonic lethality could be bypassed by superimposing a hypomorphic mutation in the Prss8 gene, encoding the glycosylphosphatidylinositol (GPI)-anchored serine protease, prostasin (CAP1/PRSS8) - a key developmental inhibitory target for HAI-2. Thus, Spint2-deficient mice homozygous for the prostasin R44Q mutation (Spint2-/-;Prss8R44Q/R44Q) were born in the expected numbers and did not exhibit any obvious developmental abnormalities at birth. However, postnatal growth in these mice was severely impaired and they all died within 4 to 7 days after birth. Interestingly, the mice displayed a CTE-like phenotype, which was characterized by widespread villous atrophy, tufted villi, near-complete loss of mucin-producing goblet cells, loss of colonic crypt structure, and bleeding into the intestinal lumen. Intestines of Spint2-/-;Prss8R44Q/R44Q mice showed altered expression of epithelial junctional proteins, including reduced levels of EpCAM, E-cadherin, occludin, and claudin-1 and -7, indicating that the loss of HAI-2 compromises intestinal epithelial barrier function. Our data show that the loss of HAI-2 in mice causes a progressive intestinal failure that at both the histological and molecular levels bears a striking resemblance to human CTE, and may provide important clues for understanding and treating this debilitating disease. Extracellular matrix degradation in physiological and pathophysiological processes Background: Research performed within the last five decades led to the identification and extensive characterization of extracellular matrix (ECM)-degrading enzymes. However, the cellular orchestration of ECM degradation and the contribution of endocytic pathways and endocytic receptors to ECM turnover is less studied and understood. We have continued our long-standing collaborative research with Niels Behrendt and Lars Engelholm, University of Copenhagen, Denmark, aimed at understanding ECM turnover. We have also initiated studies of normal and aberrant fibrin clearance in inflammation and pathologic tissue remodeling in collaboration with Niki Moutsopoulos, OIIU. Research accomplished: Tumor-associated macrophages derived from circulating inflammatory monocytes degrade collagen through cellular uptake We previously reported a key function of mannose receptor (MR)-dependent endocytic collagen degradation by M2-polarized macrophages in collagen clearance from non-neoplastic tissues. Although collagen degradation in the context of malignant tumor progression has been hypothesized to be executed directly by the malignant cells or by tumor-associated fibroblasts, this raised the possibility that macrophages could be involved in tumor-mediated extracellular matrix turnover. In collaboration with Roberto Weigert, NCI, and Lars Grntved, University of Southern Denmark by using mice with genetically-encoded fluorescently-labeled fibroblasts, tumor cell lines, and macrophages, in combination with whole mount immunostaining, we were able to visualize and quantify endocytic collagen degradation by these three principal cellular constituents of tumors in vivo. Unexpectedly, all detectable collagen uptake in tumors was observed in a subpopulation of cells identified as macrophages by their expression of CD45, CD11b, and F4/80, irrespectively of whether the tumor cells were of epithelial, mesenchymal or neural crest origin. These cells expressed MR, and internalized collagen in a MR-dependent manner. In support of this pivotal role of macrophages in tumor-associated collagen turnover, comparative whole-genome transcriptomic profiling of FACS-isolated tumor cells, tumor-associated fibroblasts, and tumor-associated macrophages showed a high collagenase expression by macrophages and fibroblasts, and a low collagenase expression by tumor cells. Furthermore, lineage-ablation studies revealed that collagen-degrading, tumor-associated macrophages mostly originated from circulating CCR2+ inflammatory monocytes. This study identifies a novel function of tumor-associated macrophages in facilitating tumor progression through endocytic collagen turnover and, furthermore, establishes blood-derived macrophages as a central cell type engaged in tumor-associated ECM destruction.